Constitutive expression of CsGI alters critical night length for flowering by changing the photo-sensitive phase of anti-florigen induction in chrysanthemum

Highlights

• Critical night length for flowering was lengthen in CsGI-OX

• CsAFT are upregulated under short day condition in CsGI-OX.

• Photo-sensitive phase inducing CsAFT was extended in CsGI-OX

• CsGI plays important roles in photoperiodic flowering in chrysanthemum.

Abstract

Chrysanthemum is a typical short day (SD) flowering plant that requires a longer night period than a critical minimum duration to successfully flower. We identified FLOWERING LOCUS T-LIKE 3 (FTL3) and ANTI-FLORIGENIC FT/TFL1 FAMILY PROTEIN (AFT) as a florigen and antiflorigen, respectively, in a wild diploid chrysanthemum (Chrysanthemum seticuspe). Expression of the genes that produce these proteins, CsFTL3 and CsAFT, is induced in the leaves under SD or a noninductive photoperiod, respectively, and the balance between them determines the progression of floral transition and anthesis. However, how CsFTL3 and CsAFT are regulated to define the critical night length for flowering in chrysanthemum is unclear. In this study, we focused on the circadian clock-related gene GIGANTEA (GI) of C. seticuspe (CsGI) and generated transgenic C. seticuspe plants overexpressing CsGI (CsGI-OX). Under a strongly inductive SD (8 L/16D) photoperiod, floral transition occurred at almost the same time in both wild-type and CsGI-OX plants. However, under a moderately inductive (12 L/12D) photoperiod, the floral transition in CsGI-OX plants was strongly suppressed, suggesting that the critical night length for flowering was lengthened for CsGI-OX plants. Under the 12 L/12D photoperiod, CsAFT was upregulated in CsGI-OX plants. Giving a night break (NB) 10 h after dusk was the most effective time to inhibit flowering in wild-type plants, while the most effective time for NB was extended to dawn (12 and 14 h after dusk) in CsGI-OX plants. In wild-type plants, a red-light pulse delivered 8 or 10 h after dusk induced maximal CsAFT expression, but the length of the time period over which CsAFT could be induced by red light was extended until subjective dawn in CsGI-OX plants. Therefore, CsGI-OX plants required a longer dark period to maintain lower levels of CsAFT, and their critical night length for flowering was thus lengthened. These results suggested that CsGI has an important role in the control of photoperiodic flowering through shaping the gate for CsAFT induction by light in chrysanthemum.

Introduction

Photoperiod is a major environmental cue for flowering. Many plant species must reproduce at the appropriate time of the year, which they achieve by measuring changes in day length and responding either to long days (LDs) or short days (SDs). Day length is measured through an endogenous circadian clock, which generates biological rhythms with approximately 24 h periods. Photoperiodic flowering is triggered when the external light signals coincide with the light-sensitive phase of the plant’s internal clock [1]. GIGANTEA (GI) was isolated as key regulator of photoperiodic flowering in Arabidopsis, a LD-flowering plant [2,3]. The circadian clock-controlled flowering pathway comprising the genes GI, CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering under inductive LD photoperiods [4]. The gene products of FT in Arabidopsis and of Heading date 3a (Hd3a) in rice (Oryza sativa), a SD-flowering plant, are proposed to be florigens that regulate flowering [[5], [6], [7]].

The plant circadian clock is controlled by a central oscillator that consist of multiple interlocked negative feedback loops. The proteins LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), and TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1) have been proposed as components of the core loop of the circadian oscillator in Arabidopsis [8]. GI physically interacts with an F-box protein, ZEITLUPE (ZTL), which targets TOC1/PRR1 for degradation and plays important roles in circadian oscillations [9]. A recent study demonstrated that GI forms a complex with HSP90 and ZTL, and acts as a molecular co-chaperone to enhance ZTL protein maturation during the light period [10]. GI also forms a complex with FKF1, a protein belonging to the same family as ZTL, and degrades CYCLING DOF FACTOR 1 (CDF1), a blue light-dependent transcriptional repressor of CO [11]. CO expression is induced by the circadian clock and CO activates FT transcription under LDs and initiates flowering.

The apparently universal function of GI in photoperiodism has been noted in many plant species [[12], [13], [14], [15], [16], [17], [18], [19], [20]]. In rice, which is a facultative SD plant, a GI orthologue (OsGI) has an important role in the control of photoperiodic flowering through the regulation of CO orthologue (Hd1) and Hd3a [12,15,21,22]. Further, rice has alternative and unique floral regulators, including Grain number, plant height and heading date 7 (Ghd7), a floral repressor, and Early heading date 1 (Ehd1), a floral promoter, which function independently of Hd1 [23,24]. The timing of the expression of both Ghd7 and Ehd1 is regulated by gating mechanisms, with the term ‘gate’ in this context referring to a light-sensitive phase set by the circadian clock. The gate for Ghd7 induction with red light opens differently depending on the photoperiod at which the plant is entrained. However, the gate for Ehd1 induction by blue light opens around dawn regardless of the photoperiod, and OsGI was found to be essential for both shaping the gate around dawn and the blue light signalling cascade [25]. In Japanese morning glory (Pharbitis nil), an obligate SD plant, the constitutive expression of a GI orthologue (PnGI-OX) suppressed flowering and the dark-induced expression of FT genes (PnFT1 and PnFT2). Interestingly, the circadian expression of PnFT1 had a longer period in plants constitutively expressing PnGI-OX, suggesting that GI has important roles in controlling the circadian expression of flowering time genes in dark-dominant plants [16].

The chrysanthemum, Chrysanthemum morifolium Ramat., is an important ornamental flower that is also a typical SD plant. Previously, we reported the presence of three FT-like genes (CsFTL1, CsFTL2, and CsFTL3) and two TFL1/CEN/BFT-like genes (CsAFT and CsTFL1) in a diploid model chrysanthemum species, C. seticuspe [[26], [27], [28]]. In this plant, expression of the CsFTL3 gene encoding florigen is induced in leaves under SDs and participates substantially in floral transition and the further development of the condensed inflorescence, called the capitulum. Expression of CsAFT, which encodes an antiflorigen, is induced in leaves under LDs or after a night break (NB), and performs an essential function in the repression of floral transition under non-inductive conditions [27]. CsTFL1 is constitutively expressed in shoot tips, regardless of the photoperiod, to inhibit floral transition [28]. The balance between the expression of FT-like genes and TFL1/CEN-like genes defines the critical day length for flowering in chrysanthemum [29].

Although both chrysanthemum and rice are classified as SD plants, the regulation of photoperiodic flowering may differ between these two species. In rice, detailed analyses of the flowering responses of wild-type and hd1-mutant plants under non-24 h light/dark cycles have suggested that a circadian rhythm set by the dawn signal is critical for day-length recognition [30]. However, in C. seticuspe, similar experiments under non-24 h atypical photoperiods and analyses of CsAFT expression have revealed that a circadian rhythm set by the dusk signal is critical for dark-time measurement, and the initiation of flowering in this species thus relies on the absolute duration of the period of darkness [27]. Although the dark-dominant flowering behaviour of chrysanthemum is very similar to that of Pharbitis [31], little is known about the molecular functions of circadian clock-related genes in these two species. We previously identified a gene showing significant homology to GI in chrysanthemum, which we named CsGI (AB733627) [32]. In the present study, we generated transgenic C. seticuspe constitutively expressing CsGI (CsGI-OX plants) and explored the effects of this gene’s constant expression on photoperiodic flowering. CsGI-OX plants showed an altered critical night length for flowering and an extended duration of the photo-sensitive phase for CsAFT induction. Our findings suggest that GI plays an important role in the control of photoperiodic flowering, probably by shaping the gate for floral regulators in dark-dominant plant species.